protein apparatus
DESCRIPTION
Protein Apparatus. Protein electrophoresis. Treat with SDS before electrophoresis Makes proteins negatively charged Performs cell lysis Partially denatures proteins Then, heat at 95 degrees to fully denature proteins Also, treat with b - mercaptoethanol to break disulfide bonds. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/1.jpg)
Protein Apparatus
![Page 2: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/2.jpg)
Protein electrophoresis
• Treat with SDS before electrophoresis– Makes proteins negatively charged– Performs cell lysis– Partially denatures proteins
• Then, heat at 95 degrees to fully denature proteins
– Also, treat with b-mercaptoethanol to break disulfide bonds
![Page 3: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/3.jpg)
Protein electrophoresis• Run vertically
• Use polyacrylamide gels
• Tris/Glycine/SDS running buffer
• Stain with Coomassie blue after electrophoresis
• Do not stain if performing Western Blot
![Page 4: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/4.jpg)
We will use premade gels: they are difficult to pour and contain toxic components
![Page 5: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/5.jpg)
Remember to wear gloves when handling gels
![Page 6: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/6.jpg)
Remove gel from box and tear side of wrapping
![Page 7: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/7.jpg)
Remove gel from protective pouch
![Page 8: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/8.jpg)
You need to pull and remove the strip at the bottom of the gel
![Page 9: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/9.jpg)
Remove the comb from the gel(after the apparatus is set up)
and wash out the wells
![Page 10: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/10.jpg)
Remove the comb from the gel
![Page 11: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/11.jpg)
Setting up the gel• Place your gel in the top
apparatus to the left– The wells face in
• After the gel is placed in, put the top apparatus into the bottom apparatus – close the locks
• You must have 2 gels: use a blank gel if you have only one gel to run
![Page 12: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/12.jpg)
Final setup of gels
![Page 13: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/13.jpg)
Make sure the wells are facing inward
![Page 14: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/14.jpg)
2 gels should be put into the apparatus
• Buffer can then be poured in between the 2 plates
• Notice the black and red electrodes
![Page 15: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/15.jpg)
Place the entire gel apparatus into the tank
![Page 16: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/16.jpg)
You may want to use longer/thinner tips to load the gel
![Page 17: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/17.jpg)
Remember, the wells are in the back of the gel
• Make sure you find the wells
• Careful place the tip as far down as possible
• Load the well
![Page 18: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/18.jpg)
Another picture of gel loading
![Page 19: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/19.jpg)
The final step
• Put the cover on in the correct orientation
• Red to red and black to black
![Page 20: Protein Apparatus](https://reader036.vdocuments.net/reader036/viewer/2022062813/56816487550346895dd65f87/html5/thumbnails/20.jpg)
Gel after staining with Coomassie Blue
• Standards Measured in kD
• Fig. 7.12
• 10 kD – 250 kD